Human calcium binding domain biosensors

ABSTRACT

This invention provides biosensors, cell models, and methods of their use for monitoring ionic or voltage responses to contact with bioactive agents. Biosensors can include targeting domains, sensing domains and reporting domains. Biosensors can be introduced into cells reprogrammed to represent experimental or pathologic cells of interest. Model cells expressing the biosensors can be contacted with putative bioactive agents to determine possible activities.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and benefit of a prior U.S.Provisional Application No. 61/873,950, Human Cellular Models withBiosensors, by Angela Huang, filed Sep. 5, 2013. The full disclosure ofthe prior application is incorporated herein by reference.

FIELD OF THE INVENTION

The present inventions are in the field of methods and compositions foractive agent screening, monitoring of electrical impulses, and formonitoring signal transduction. The inventions present compositions fordetection of specific bioactivity, e.g., in targeted regions ofrepresentative host cells. Methods include transformation ofreprogrammed human cells with targeted sensor constructs specific for,e.g., detection of changes in the ionic environment or voltage potentialin a cell.

BACKGROUND OF THE INVENTION

Detection of voltage potentials and changes to the internal ionicenvironment of cells can be useful in monitoring bioactivities of cells.For example, many cells experience significant changes in internalcalcium ion (Ca²⁺) concentration in response to binding of a ligand to aG-protein receptor. In another aspect, certain cells experience largechanges in voltage potential across membranes, e.g., in response tocontact with neurotransmitters at a synapse. Such cellular changes areresponsible for important functions in cells and can be indicative ofthe health, function, or development processes of the cells.

Genetically encoded calcium indicators (GECI) have been developed toallow general monitoring of the Ca²⁺ concentration in cells. See, e.g.,Looger (U.S. 2012/0034691), wherein a calmodulin peptide sequence iscombined in a construct with a green fluorescent protein (GFP) reporter.Conformational changes in the calmodulin peptide in response to Ca²⁺binding changes the efficiency of the GFP fluorescence, resulting in achange in the emissions profile and intensity. Cells transformed withthe GECI can be monitored generally for changes in internal Ca²⁺concentration, e.g., in response to signaling ligands or inhibitors.Also see Griesbeck (U.S. 2009/0035788) wherein a FRET donor and acceptorare separated by a troponin peptide sequence, resulting in afluorescence change on binding of Ca²⁺. However, such GECIs are limitedin their resolution of signal, limited in ability to penetratemulti-cell/3D structures, and in the range of available applicable celltypes. Typically, the old art systems are directed to two dimensionalmicroscopic detection of signals from a cell monolayer.

In many cases, signal transduction studies are carried out in cell typesthat are not representative of the actual cells of interest. Forexample, researchers may be limited to studying signaling agents andpotential therapeutics in rodents or immortal cell lines in vitro, whichoften provide results not repeated in human cells, or clinical patients.For example, researchers can create host cells for study by introductionof oncogenes to primary cell lines, e.g., with differentiation to a celltype of choice. However, after this process history, the cells cannot berelied on to respond normally on contact with bioactive agents. Weiss(U.S. Pat. No. 7,101,709) discloses methods of preparing multipotentmouse neural stem cells. The cells could be differentiated andtransplanted to somewhat immunoprivileged CNS locations. Again, thecells would be non-representative for many studies, and signal detectionis limited to immunochemical means.

In view of the above, a need exists for model cell systemsrepresentative of cells and tissues existing in live animal systems ofinterest. It would be desirable to have sensor peptide constructs thatcan be targeted to specific intracellular locations. Benefits could berealized if systems were available allowing three dimensional signaldetection in mock tissues of representative cells in vitro. The presentinvention provides these and other features that will be apparent uponreview of the following.

SUMMARY OF THE INVENTION

The present biosensors combine complementary features to detectintracellular changes associated with signaling in vivo. The presentbiosensors find utility in a variety of contexts. For example, thebiosensors in human iPSCs-derived cell types can function to image 2D or3D cellular models of in cells expressing certain pathologies. Suchmodels can be useful in screening and evaluation of candidate drugcompounds for chemical, biologic, therapeutic, and toxicologicaleffects.

The synthetic and genetically encoded biosensors can function asreporters of calcium (Ca²⁺) concentrations in human cells. Thebiosensors can have structures targeting cellular compartments, e.g.,such as the nucleus, cytoplasm, plasma, certain membrane surfaces,and/or the like. The biosensors can optionally function as reporters ofcellular voltage changes in human cell compartments. For example,voltage sensors can evaluate and report fluctuations in membranepotentials due to differentials or changes in sodium (Na⁺) and potassium(K⁺) concentrations.

The biosensors can be configured to function in a cell-type specificmanner. For example, biosensors can be genetically modified to containpromoter sequences specific to certain cell types, e.g., dopaminergicneurons, GABAergic neurons, astrocytes, cardiomyocytes, immortalizedhuman cancer cell lines, HSCs, NPCs, human cells in general and/or MSCs.

In one embodiment, the biosensor is a voltage sensor. The voltage sensorcan include, e.g., a peptide construct comprising a transmembranedomain, voltage sensitive domain, and a reporter domain. For example,the voltage sensor construct can include a transmembrane domain adaptedto integrate into a membrane of a human cell, a voltage sensing domainsensitive to H⁺, Na⁺ and/or K⁺ concentration, and a fluorescent reporterdomain. The fluorescent reporter can be adapted to fluoresce atwavelengths in the range from 500 nm to 750 nm. The biosensor can beadapted to change conformation (e.g., in the voltage sensing domain) inresponse to local voltage potentials, resulting in the fluorescentreporter domain changing fluorescent emission characteristics. In manyembodiments, the transmembrane domain and voltage sensing domain aredifferent domains (e.g., having different non-homologous sequences, orbeing derived from different parent peptides).

In certain embodiments of the voltage sensor system, the voltage sensorpeptide includes a voltage sensing domain comprising a sequence at least70%, 80%, 90%, 95%, 98%, or 99% identical to:

(SEQ ID NO: 1) MSSVRYEQREEPSMVNGNFGNTEEKVEIDGDVTAPPKAAPRKSESVKKVHWNDVDQGPNGKSEVEEEERIDIPEISGLWWGENEHGVDDGRMEIPATWWNKLRKVISPFVMSFGFRVFGVVLIIVDFVLVIVDLSVTDKSSGATTAISSISLAISFFFLIDIILHIFVEGFSQYFSSKLNIFDAAIVIVTLLVTLVYTVLDAFTDFSGATNIPRMVNFLRTLRIIILVRIIILVRILRLASQKTISQN.In preferred embodiments, the voltage sensor peptide sequence retainsI123, R220, R226, R229, R235 and/or R238 residues. In a more preferredembodiment, the voltage sensor domain peptide wherein the sequenceretains at least R235 and I123.

In some embodiments of the voltage sensor system, the fluorescentreporter domain peptide includes a fluorescent domain comprising asequence at least 70%, 80%, 90%, 95%, 98%, or 99% identical to:

(SEQ ID NO: 2) MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHQFKCTGEGEGRPYEAFQTAKLKVTKGGPLPFAWDILSPQFMYGSRAFIKHPAGIPDFFKQSFPEGFTWER VTRYEDGGVVTVMQDTSLED.

In certain embodiments, the voltage sensor peptide construct includesthe peptide sequences at least 70%, 80%, 90%, 95%, 98%, or 99% identicalto each of SEQ ID NO: 1 and SEQ ID NO: 2.

Another inventive aspect includes a nucleic acid construct encoding anyof the voltage sensors described herein. Further, the nucleic acidconstruct can comprise a tag sequence selected from the group consistingof: a nucleus localization signal (NLS) tag, a mitochondriallocalization tag, and a ciliary tag. Further, the nucleic acid constructcan “ ” include the NLS tag comprising at least 80% identity to thepeptide sequence:

(SEQ ID NO: 3) DPKKKRKV.

The inventive contributions of the present application include a humancell comprising the voltage sensor described herein. In preferredembodiments, the human cell is an iPSC derived cell. For example, thecell can be derived from induction of a fibroblast or a blood cell to apluripotent or immortal status. In many cases, the cell is derived froma human patient derived cell type.

The biosensors of the invention include calcium sensor. For example, acalcium sensor peptide construct can include a calcium binding domain, aEF-hand troponin-like binding domain, and fluorescent reporter domain.Often the fluorescent reporter is adapted to emit at wavelengths in therange from 500 nm to 750 nm. The fluorescent reporter domain can beadapted to change fluorescent emissions characteristics, e.g., inresponse to conformational changes in the EF-hand troponin-like domain.

The calcium sensor peptide construct can include a calcium bindingdomain comprising a calmodulin peptide sequence. For example, thecalcium sensor peptide construct can include a calcium binding domain isat least 70%, 80%, 90%, 95%, 98%, or 99% identical to:

(SEQ ID NO: 4) EFRASFNHFDRDHSGTLGPEEFKACLISLDHMVLLTTKELGTVMRSLGQNPTEAELQDMINEVDADGDGTFDFPEFLTMMARKMMNDTDSEEEGVQGTSEEEELANCFRIFDKDANGFIDEELGEILRATGEHVTEEDIEDLMKDSDKNN GRIDFGEKLTDEEV.

The calcium sensor peptide construct can include a EFhand binding domainis at least 70%, 80%, 90%, 95%, 98%, or 99% identical to:

(SEQ ID NO: 5) FKEAFSLFDKDGDGTITTKELGTVMRSL-ELDAIIEEVDEDGSGTIDFEEFLVMMVRQ.

The calcium sensor peptide construct can include a fluorescent reporterdomain is at least 70%, 80%, 90%, 95%, 98%, or 99% identical to:

(SEQ ID NO: 6) MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHQFKCTGEGEGRPYEAFQTAKLKVTKGGPLPFAWDILSPQFMYGSRAFIKHPAGIPDFFKQSFPEGFTWER VTRYEDGGVVTVMQDTSLED.

The calcium sensor peptide construct can includes the peptide sequencesat least 70%, 80%, 90%, 95%, 98%, or 99% identical to each of SEQ ID NO:4 and SEQ ID NO: 5.

Another inventive aspect described herein includes nucleic acidconstructs encoding any of the calcium sensors described herein.Further, the nucleic acid construct can comprise a tag sequence. Forexample, the tag sequence can include a targeting sequence, such as aNLS tag, a lipid membrane tag, an ER tag, a golgi tag, an endosome tag,and/or a ciliary tag.

The inventive contributions of the present application include a humancell comprising any calcium sensor described herein. In preferredembodiments, the human cell is an iPSC derived cell. For example, thecell can be derived from a fibroblast or a blood cell.

The inventions include methods of reprogramming and monitoring cells.For example, in a method of reprogramming fibroblasts can includetransforming the fibroblasts with one or more constructs comprising ahuman clock gene and human Bmal1/2/3/4 genes having E-box promoters,synchronizing the circadian rhythm of the fibroblasts, modifyingtranscriptional regulatory control of the fibroblasts (therebyconverting them into inducible pluripotent stem cells), andreprogramming the stem cells into inducible neurons (iN), glial cells(astrocytes included, iG), inducible pluripotent stem cells (iPSCs), orinducible neural progenitor cells (iNPCs). In many of the methods forreprogramming using circadian rhythms of human cell types, thetransforming construct comprises a nucleic acid encoding a voltagesensor described herein or calcium sensor described herein.

The methods can include, e.g., modifying transcriptional control byproviding specific transcription factors suitable for a lineage of thefibroblasts. The transcription factors can include the factors specificfor a cellular lineage of the fibroblasts modified to include acircadian regulatory element (E-box promoters, an artificial E-box-likepromoter sequence tag, a chemical agent that alters or synchronizescircadian rhythms cycles, or a synthetic transcriptional enhancerelement) for reprogramming using circadian rhythms of human cell types.

The cellular composition can further include the co-culturing iN cellsand iG cells to create a 3D model on a scaffold in vitro. Also, humancancer cell lines and cancer stem cells can be cultured in 3D spheroidmanners using standard culturing hardware and conditions. Biosensorconstructs of the invention can be used to image the resultant tissuesand monitor changes in the cell voltage potentials and/or calciumlevels, e.g., in real time.

Heme-binding and ATP-binding biosensors can be designed using similarlogic as the calcium and voltage biosensors described herein. Bothbiosensors are genetically encoded, with specific domains for binding toheme molecules or ATP, respectively. These biosensors are attached to afluorescent reporter (600 nm-700 nm range), whose intensities aremodulated due to conformational changes due to domain binding. Inalternatives to a single fluorescent or intensiometric reporter, thebiosensor domains can be attached to a pair of FRET fluorescentreporters of various wavelengths and become a FRET sensor.

The present inventive methods include compositions and techniques forscreening agents that influence the calcium or voltage potential ofcells. For example, methods for screening active agents can includetransforming one or more cells with a nucleic acid encoding a voltagesensor or calcium sensor, expressing the voltage sensor or calciumsensor in the cells, contacting the cells with candidate active agents,and detecting a change in florescence of the voltage sensor or calciumsensor in response to the agent. Typical agents can include, e.g.,members of a small molecule chemical library. For example, the agentscan be reviewed for an activity resulting from interactions with GPCR, amembrane channel, a receptor, and/or associated signaling pathways.

It is further envisioned that the methods are useful in imaging livecells in low, medium, and/or high throughput assay formats. For example,cells in 3D arrays, transformed and expressing biosensor constructs ofthe invention can be viewed in real time, e.g., using microscopicimaging systems. Optionally, biosensor cells in surface or suspensionculture can be monitored using confocal imaging, FACS sorters, CCD videoimaging, and/or the like.

3D human cancer cell models have been used as an improved predictor oftumor responses to drug candidates using library screening highthroughput screening (HTS) methods. Cells used for such studies aren'tlimited to immortalized cancer cell lines. Cancer stem cells, primarytissue cells, and 3D printed tissue types can be used for screening.

DEFINITIONS

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular devices orbiological systems, which can, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting. As used in this specification and the appended claims, thesingular forms “a”, “an” and “the” include plural referents unless thecontent clearly dictates otherwise. Thus, for example, reference to “asurface” includes a combination of two or more surfaces; reference to“bacteria” includes mixtures of bacteria, and the like.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein. In describing and claimingthe present invention, the following terminology will be used inaccordance with the definitions set out below.

iPSC refers to inducible pluripotent stem cells; iN refers to inducibleneurons; iG refers to inducible glial cells (including astrocytes); andiNPCs inducible neural progenitor cells.

Near-Infrared refers to light wavelengths ranging from about 600 nm toabout 1400 nm.

The term “conservative variant” includes modifications of givensequences that result in conserved function. For example, in the contextof nucleic acids, owing to the degeneracy of the genetic code, “silentsubstitutions” (i.e., substitutions in a nucleic acid sequence which donot result in an alteration in an encoded polypeptide) are an impliedfeature of every nucleic acid sequence which encodes an amino acid.

Similarly, conservative variants in the context of peptide sequences canbe expected to retain function. For example, Guo, “Protein Tolerance toRandom Amino Acid Change”, (PNAS 101:9205-10; 2004), demonstrates thatone of skill can modify peptides successfully even “without detailedknowledge of the ways in which a protein's structure relates to itsfunctional usefulness . . . ” Guo finds only 25% of random mutationslead to substantial loss of activity. Guo extensively discusses how oneof skill can take into consideration active site location, alphahelices, beta sheets, hydrophobic interactions, turns and loops,conserved sites and the like to intelligently avoid loss of activity,e.g., by substitution avoidance at key positions or with conservativeamino acid substitutions. Further, Guo states that his “database can bea valuable resource for predicting the effects of mutations on proteinfunction . . . .” Substitutions to known structures are predictable andin possession the of those having the structural information. Therefore,conservative amino acid substitutions, in which one or a few amino acidsin an amino acid sequence are substituted with different amino acidswith highly similar properties, are also readily identified as beinghighly similar to a disclosed constructs, and expected to retainfunction. One of skill will recognize that individual substitutions,deletions, or additions which alter, add or delete a single amino acidor a small percentage of amino acids (typically less than 5%, moretypically less than 4%, 2% or 1%) in an encoded sequence are“conservatively modified variations” where the alterations result in thedeletion of an amino acid, addition of an amino acid, or substitution ofan amino acid with a chemically similar amino acid. Thus, “conservativevariations” of a listed polypeptide sequence of the present inventioninclude substitutions of a small percentage, typically less than 5%,more typically less than 2% or 1%, of the amino acids of the polypeptidesequence, with a conservatively selected amino acid of the sameconservative substitution group.

TABLE 1 Conservative Substitution Groups 1 Alanine (A) Serine (S)Threonine (T) 2 Aspartic acid (D) Glutamic acid (E) 3 Asparagine (N)Glutamine (Q) 4 Arginine (R) Lysine (K) 5 Isoleucine (I) Leucine (L)Methionine (M) Valine (V) 6 Phenylalanine (F) Tyrosine (Y) Tryptophan(W)

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a chart wherein TempoCal™ has been incorporated into humancells and stimulated with histamine (20 μM), carbachol (20 μM), andionomycin (20 μM) for an average response 9-12 fold over control.Histamine: a known substance that stimulates intracellular calciumincreases. Carbachol: a known medication to treat glaucoma; ananti-apoptosis agent, aCHR agonist, and TNF-alpha inhibitor. Ionomycin:an ionophore used in research to raise intracellular levels of calcium.

FIG. 2 shows a chart wherein TempoMito™-containing cells have beenstimulated with histamine (20 μM), and carboxyamidotriazole (20 μM).Histamine: a known substance that stimulates intracellular calciumincreases. Carboxyaminoimidazole (CAI): an orally-active agent,non-voltage-operated calcium channel blocker.

FIG. 3 shows TempoVol™-incorporated cells respond to pH 3, 0.1M CaCl₂,EDTA, and Ca²⁺ Mg²⁺ Free solutions.

FIG. 4 shows TempoCal™, TempoMito™, and TempoVol™ cellular responsesfrom a single 2.5 mM CaCl₂ stimulation.

FIG. 5 shows TempoVol™: cellular responses to pH3, 1.0 mM CaCl₂, 10 mMCaCl₂, and 10 mM EDTA.

FIG. 6 shows TempoMito™ responses using U2-OS human osteoblastoma cells.

FIG. 7 shows Tempo's iPS derived human neural progenitor cells.Immunohistochemistry employs biomarker vimentin antibodies. Humanvimentin is an intermediate filament type and is expressed by neuralprogenitor/stem cells of the central nervous system.

FIG. 8 shows Tempo's iPS derived human neural progenitor cells in alight transmission microscopic field.

FIG. 9 shows TempoCal™ biosensor expressed in Tempo's iPS derived humanneural progenitor cells.

FIG. 10 shows Tempo's iPS derived human astrocyte cells in a lighttransmission view.

FIG. 11 shows Tempo's iPS derived human astrocyte cells.Immunohistochemistry employs biomarker GFAP antibodies. GFP (glialfilament protein) is a class-III intermediate filament and a cellspecific marker that distinguishes astrocytes from other glial cellsduring the development of the central nervous system.

FIG. 12 shows the TempoCal™ biosensor expressed in Tempo's iPS derivedhuman astrocyte cells.

FIG. 13 shows the TempoMito™ biosensor expressed in Tempo's iPS derivedhuman astrocyte cells.

FIG. 14 shows the TempoMito™ biosensor expressed in Tempo's iPS derivedhuman neural progenitor cells.

FIG. 15 shows the TempoCal™ biosensor expressed in Tempo's proprietaryneuroepithelial cells.

FIG. 16 shows the TempoMito™ biosensor expressed in Tempo's proprietaryneuroepithelial cells.

FIG. 17 shows Tempo's iPS derived human oligodendrocyte progenitorcells. Immunohistochemistry employed biomarker O4 antibodies.

FIG. 18 shows Tempo's iPS derived human oligodendrocyte progenitor cellsinduced with T3 hormone (70 ng/ml) after 6 days Immunohistochemistryemploys biomarker myelin basic protein (MBP) antibodies.

FIG. 19 shows the TempoVol™ biosensor expressed in Tempo's proprietaryneuroepithelial cells.

FIG. 20 shows Tempo's iPS derived human dopaminergic neurons.Immunohistochemistry employs biomarker Thymine Hydroxylase (TH)antibodies.

FIG. 21 shows Tempo's immortalized neuroepithelial cells tested as a 3Dspheroid model using Lipidure-coated 96-well U-shaped plates. Diameterof spheroid shown=250 μm.

DETAILED DESCRIPTION

The present invention includes biosensor constructs, reprogrammingmethods to prepare cells useful in receiving the constructs, and assaymethods employing the biosensors, e.g., to detect the presence of activeagents or signals in living cells.

The biosensors generally include various complementary domain structuresworking together to sense a voltage or ion at a particular intracellularlocation, and to provide a distinct signal correlated to changes in theparameter. In a particularly useful embodiment, the biosensors areexpressed in cells representative of a particular species (e.g., human),cell type of interest, or in a cell expressing a pathology of interest.Cells expressing the biosensors can be exposed to conditions or agentsand monitored for signals indicating cell responses.

Biosensors Generally.

Biosensors of the present invention are generally engineered to includecomponents specialized in providing, e.g., location, sensing, andreporting. In expression of a sensor nucleic acid construct, the peptideproduct can be positioned within the cell, e.g., by chaperone or transitsequences, hydrophobic affinities, or ligand/receptor interactions. Thesensing domains typically change conformation in response to a changedintracellular condition or binding of a signal ion or molecule.Reporting domains typically present a detectable signal that changes inresponse to conformational changes in the associated sensing domain.

The intracellular location of a biosensor can optionally be controlledby a targeting domain or “tag”. In some biosensors, intracellularlocation is generalized or passively determined. For example, the sensormay be generally dispersed throughout the nucleus and/or cytoplasm. Inother cases, the function or specificity of the biosensor signal maydepend on localization at a particular intracellular membrane ororganelle. Examples biosensor tags include hydrophobic peptides directedto interact with membranes, ionic peptides directed to disperse incytoplasm, chaperone sequences directing the biosensor to a particularcompartment, and/or ligands directed to receptors. Tags useful in thestudy of intracellular signaling and in agent screening assays caninclude, e.g., NLS tags, lipid membrane tag, endoplasmic reticulum (ER)tags, golgi tags, endosome tags, ciliary tags, and/or the like.

Sensing domains in the biosensors change conformation in response to achanged physical condition, binding of a ligand, or change in an ionicenvironment. The conformational changes can, e.g., cause conformationalchanges in an associated reporter domain, or reposition the reporterdomain to enhance or diminish the signal. Sensing domains can changeconformation in response to binding of a peptide, binding of a nucleicacid, interaction with a protease, interaction with a phosphatase,changes in pH, changes in ionic strength, changes in a voltagepotential, and/or the like.

Reporter domains of the biosensors can be of any appropriate type knownin the art. However, in preferred embodiments, the reporters compriseone or more peptide domains, e.g., so they can be easily employed in invivo systems. Typically the peptide reporter domain provides a specificfluorescent emission in response to a specific interrogating excitationwavelength of light. In the context of sensor domain conformationalchanges, FRET strategies can be effective, e.g., wherein the biosensorconstruct comprises paired donor/acceptor peptide pairs. In certainembodiments, the reporter domain is adapted to provide an emission inthe red to near-infrared spectrum, e.g., to allow the signal to pass andbe detected in three dimensions through cells or tissue layers. Forexample, relatively penetrating reporter emission signals can range from500 nm to 1400 nm, 550 nm to 900 nm, 600 nm to 850 nm, 650 nm to 800 nm,or about 700 nm. In an exemplary embodiment, the reporter domainincludes fluorescence enhancing amino acids, such as tryptophan,tyrosine, phenylalanine, which contribute to their intrinsicfluorescence of the domain. Optionally, the reporter domain can includeregions naturally modified post-translationally to provide the desiredlong wavelength emissions. For example, a domain that emits in the redregions can be a modified green fluorescent protein where thefluorophore originates from an internal serine-tyrosine-glycine sequencewhich is modified to a 4-(p-hydroxybenzylidene)-imidazolidin-5-onestructure.

Biosensor constructs can include, e.g., two, three, or four of the abovedescribed domain types, in any of a variety of functionalconfigurations. For example, a sensor domain and reporter domain in anyorder can constitute a biosensor. Often, the biosensors of the inventioninclude at least three domains, e.g., a targeting domain, a sensordomain, and a reporting domain. The three domains can be in any order,but typically the targeting domain is on one end of the construct, thesensor in the middle and the reporter on the second end. In certainconfigurations, the biosensor construct can be in the order(C-terminal/N-terminal or N-terminal/C-terminal) of: sensor/reporter;targeting/sensor/reporter; targeting/reporter/sensor;targeting/reporter1/sensor/reporter2 (e.g., FRET);reporter1/sensor/reporter2; sensor/reporter1/reporter2; andtargeting/sensor/reporter1/reporter2.

Typically, the domains are linked together in a commonly translatedconstruct of a single linear peptide. Optionally, the constructs caninclude one or more domains not in the same peptide chain as anotherdomain. For example, separate domains may be associated in anon-covalent interaction, such as a hydrophobic interaction, achelation, a ligand/receptor interaction, an antibody/antigeninteraction, and/or the like.

In some cases, a single domain may have more than one function. Forexample, a sensor domain may also have a structure functioning as atargeting domain. In one embodiment, a domain may have a series oftransmembrane domains, acting as both as a sensor (e.g., ligandresponsive ion channel) and a membrane specific targeting domain. Inanother aspect, a sensor domain could also include a reporter function,e.g., acting as a donor or quencher member of a FRET pair with aseparate reporter domain.

Voltage Sensing Biosensors.

Voltage sensors of the invention include many of the aspects describedabove, wherein the sensor domain is responsive to changes in voltagepotential. For example, the voltage sensor may be sensitive toelectrostatic influences in the local environment, e.g., a voltagedifferential across a membrane to which it is bound. Optionally thevoltage sensor is sensitive to the local ionic environment. In livingcells, the voltage potential is usually related to the absoluteconcentration of H⁺, Ca²⁺, K⁺ and/or Na⁺, or a concentrationdifferential of these ions across a membrane. Therefore, voltage sensordomains typically include domains sensitive to electrostatic forces, H⁺,Ca²⁺, K⁺ and/or Na⁺ concentration. Such sensors are particularly usefulin association with relatively electroactive cells such as muscle cells,heart cells, and nerve cells.

Importantly, such voltage sensors can be configured to target and reportthe ion/voltage status specifically at intracellular locations. Forexample, the construct can include a targeting domain directing theconstruct to a cellular compartment or surface such as the nucleus,sacroplasma, plasma membrane, nerve axon, cilia, or synapse. Thus, notonly can signal transduction be monitored in a cell generally, but morespecific voltage differential effects can be visualized at thesub-cellular level.

Typical voltage sensitive biosensors of the invention include atransmembrane domain, voltage sensing domain, and a reporter domain. Thetransmembrane domain may act as a targeting domain and/or a voltagesensing domain, in some instances. In one embodiment, the voltage sensorhas the domains in the order transmembrane/sensor/reporter. In preferredembodiments, the reporter is a fluorescent peptide, e.g., emitting inthe red to near-infrared range.

A preferred voltage biosensor construct includes a combination of an ionchannel transmembrane peptide, an ion channel voltage sensing peptidedomain, and a fluorescent peptide. In an exemplary embodiment, thevoltage sensor includes a modified transmembrane peptide, a modifiedvoltage, and a fluorescent protein modified to provide red emissions.The domains of the constructs are typically configured from acombination of bioinformatics/database sequences as modifiedevolutionary mutagenesis.

In a particular voltage sensing domain the sequence comprises:

(SEQ ID NO: 1) MSSVRYEQREEPSMVNGNFGNTEEKVEIDGDVTAPPKAAPRKSESVKKVHWNDVDQGPNGKSEVEEEERIDIPEISGLWWGENEHGGDDGRMEVPATWWNKLRKVISPFVMSFGFRVFGVVLIIVDFVLVIVDLSVTDKSSGATTAISSISLAISFFFLIDIILHIFVEGFSQYFSSKLNIFDAAIVIVTLLVTLVYTVLDAFTDFSGATNIPRMVNFLRTLRIIRLVRIIILVRILRLASQKTISQN.

Conservative variations of the sequence would be expected to retainsubstantial useful function. The present voltage sensor domains includepeptides comprising sequences at least 70%, 80%, 85%, 90%, 95%, 98%, or99% identical to SEQ ID NO: 1. Function is expected to be best preservedwith conservative substitutions and if the sequence retains at least 1,2, 3, 4, 5, or all 6 of the following amino acid residues: I123, R220,R226, R229, R235, or R238. In many embodiments, preservation of R235 andI123 can be particularly useful in retaining optimal voltage sensorfunction.

In a particular reporter domain for the voltage sensor peptide, thesequence comprises:

(SEQ ID NO: 2) MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHQFKCTGEGEGRPYEAFQTAKLKVTKGGPLPFAWDILSPQFMYGSRAFIKHPAGIPDFFKQSFPEGFTWER VTRYEDGGVVTVMQDTSLED.Conservative variations of the sequence would be expected to retainsubstantial useful function. The present fluorescent reporter sensordomains include peptides comprising sequences at least 70%, 80%, 85%,90%, 95%, 98%, or 99% identical to SEQ ID NO: 2.

Preferred voltage sensing biosensors of the invention include both thevoltage sensor of SEQ ID NO: 1 and the fluorescent reporter of SEQ IDNO: 2, and/or their conservative variants, as discussed above.

Calcium Ion Sensing Biosensors.

Calcium sensor constructs of the invention include many of the aspectsdescribed above for biosensors generally, but the sensor domain isresponsive to changes in calcium ion levels. For example, the calciumsensor will typically bind Ca²⁺ with a certain affinity and changeconformation to some degree depending on the local Ca²⁺ concentration.Ca²⁺ can vary dramatically depending on cell type, and according to theinfluence of induced signals. For example, it can be informative tomonitor muscle cells, nerve cells, cells responding to g-proteincontrolled signals, cells undergoing apoptosis, and/or the like. Inliving cells, Ca²⁺ levels often vary with intracellular locations. Thepresent calcium sensors can include targeting domains directing thesensors to any intracellular compartment of membrane, such as, e.g., avacuole, the nucleus, cytoplasm, synapse, endoplasmic reticulum, and/orthe like.

Calcium sensor domains are typically peptides homologous to portions ofone or more calcium binding proteins. For example, a calcium bindingdomain can have a sequence similar to an evolutionary sequence found incalmodulin, calexcitin, parvalbumin, S100 proteins, calcineurin, and/orthe like. All that is necessary for the sensor role is that the peptide,or peptide fragment, changes conformation with changes in calciumconcentration. The change in conformation will translocate the reportergroup and typically change the emission profile or intensity. Modernprotein engineering techniques can be used to engineer enhancementscausing the translocation of the reporter to be enhanced or quenched,e.g., by induced contact or induced conformational changes in thereporter itself.

A preferred calcium biosensor construct includes a combination of atandem array of calcium binding domains (namely, EFhand domains) thatinclude calmodulin and troponin motifs, and a fluorescent peptide. In anexemplary embodiment, the calcium sensor includes a modified calciumbinding domain from calmodulin, troponin, and a fluorescent proteinmodified to provide red emissions. The domains of the constructs aretypically configured from a combination of bioinformatics/databasesequences as modified evolutionary mutagenesis.

In a particular calcium binding domain the sequence comprises:

(SEQ ID NO: 4) EFRASFNHFDRDHSGTLGPEEFKACLISLDHMVLLTTKELGTVMRSLGQNPTEAELQDMINEVDADGDGTFDFPEFLTMMARKMMNDTDSEEEGVQGTSEEEELANCFRIFDKDANGFIDEELGEILRATGEHVTEEDIED LMKDSDKNNGRIDFGEKLTDEEV.Conservative variations of the sequence would be expected to retainsubstantial useful function. The present calcium binding domains includepeptides comprising sequences at least 70%, 80%, 85%, 90%, 95%, 98%, or99% identical to SEQ ID NO: 4.

In a particular EFhand domain for the calcium sensor peptide, thesequence comprises:

(SEQ ID NO: 5) FKEAFSLFDKDGDGTITTKELGTVMRSL--ELDAIIEEVDEDGSGTIDFEEFLVMMVRQ.Conservative variations of the sequence would be expected to retainsubstantial useful function. The present EFhand domains include peptidescomprising sequences at least 70%, 80%, 85%, 90%, 95%, 98%, or 99%identical to SEQ ID NO: 5.

In a particular reporter domain for the calcium sensor peptide, thesequence comprises:

(SEQ ID NO: 6) MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHQFKCTGEGEGRPYEAFQTAKLKVTKGGPLPFAWDILSPQFMYGSRAFIKHPAGIPDFFKQSFPEGFTWERVTRYEDGGVVTVMQDTSLED.Conservative variations of the sequence would be expected to retainsubstantial useful function. The present fluorescent reporter sensordomains include peptides comprising sequences at least 70%, 80%, 85%,90%, 95%, 98%, or 99% identical to SEQ ID NO: 6.

Preferred calcium sensing biosensors of the invention include one, two,three or more of the domains identified in SEQ ID NOs: 4, 5, 6, and 7,and/or their conservative variants, as discussed above.

Biosensor Constructs in Human Cells.

The biosensors described herein can be incorporated into cells tomonitor voltage and ion conditions within the cells. For example,nucleic acid constructs encoding the biosensor peptide domains can betransformed or transfected into eukaryotic cells for expression, e.g.,using appropriate promoters, as is known in the art. The cells arepreferably human, providing the benefit of a reliable host cell model,e.g., for study of human signal transduction and disease states. Thecells are preferably stem cells or cells differentiated to a particularcell type of interest.

The nucleic acid construct can encode any biosensor described herein.For example, the construct can encode a combination of a voltage sensordomain, calcium sensor domain, targeting domain, and/or reporter domain.In one embodiment, the nucleic acid construct includes transientexpression vector components directing expression of peptide chainscomprising a combination of domains providing a functional biosensor.For example, the nucleic acid can express a single peptide chaincomprising a combination of calcium binding domain, troponin domain, andreporter domain. In another example, the nucleic acid construct canencode a peptide comprising at least a combination of a transmembranedomain, a voltage sensing domain, and reporter domain.

In other embodiments of the nucleic acid expression constructs, anexpression vector includes sequences encoding peptides of any of SEQ IDNOs: 1, 2, 3, 4, 5, 6, and/or 7. In certain embodiments functioning asvoltage biosensors, the vector can include a combination of nucleic acidsequences encoding the peptides of SEQ ID NOs: 1 and 2, e.g., along witha sequence encoding a transmembrane domain, such as a G-protein domainor an ion channel domain. Such a construct can encode functional peptidesequences at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% identical to thesequence of SEQ ID No: 1 and/or SEQ ID NO: 2. In preferred embodiments,the construct encodes a homologue of SEQ ID NO: 1 retaining at least theamino acids at I123, R220, R226, R229, R235, and/or R238. In manyembodiments, preservation of R235 and I123 can be particularly useful inretaining optimal voltage sensor function. In other embodimentsfunctioning as calcium sensitive biosensors, the vector can include acombination of nucleic acid sequences encoding the peptides of SEQ IDNOs: 4, 5, 6, and/or 7. Such a construct can encode functional peptidesequences at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% identical to thesequence of SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6, and/or SEQ ID NO:7.

Useful nucleic acid constructs of the above expression vectors caninclude additional sequences encoding targeting tags, such as, e.g., aNLS tag, a lipid membrane tag, an ER tag, a golgi tag, an endosome tag,a mitochondrial tag, and/or a ciliary tag. For example, the targetingtag can include a sequence at least 90% identical to a sequence encodingthe peptide tag sequence of SEQ ID NO: 3.

The present inventions include cells comprising the biosensors discussedherein. For example, nucleic acid constructs coding the biosensorpeptides can be transduced or transfected into eukaryotic cells ofchoice. In preferred embodiments, the cells originate from a mammal,most preferably from a human. In many embodiments, the cell is animmortalized stem cell, or a cell fully or partially differentiated froma stem cell.

In the context of the present biosensors, cells can be initially derivedfrom human patient samples. The advantage of such cells is that they canprovide very representative responses to active agents and changedconditions for that patient. Thus, such cell models are more likely toprovide information on the modes of action or efficacy of a candidatetherapeutic for that patient. For example, such cell models can aid inthe identification of custom tailored treatment for patients withcertain disease states, such as autoimmune diseases, neurologicaldiseases, cancer, diabetes, or pathologies from genetic flaws.

Alternately, the models can employ cells harboring a particularnon-endogenous gene of interest, introduced by genetic engineeringtechniques. For example, the gene of interest can encode a receptormolecule, a G-protein coupled receptor, or an ion channel of interest.Typically, the host cell is a cell most representative of the cell typeof interest in the research. Such model cells can be useful in providingmore representative results, e.g., in monitoring a signal transductionor in screening prospective agents active in the modulation of the geneof interest.

Cellular models can be derived from inducible cells available fromprimary culture of cells from living animals. For example, fibroblastsor undifferentiated cells from circulating blood can be induced toprovide pluripotent cells. It is notable that epigenetic processes canplay a key role in directing the status of a cell to stem cell,progenitor cell, or mature cell. In Lister (Nature 471 (7336): 68-73,2011), aberrant epigenomic programming was found capable of inducing avariety of different pluripotent stem cells (iPSCs). Female lungfibroblasts, adipose cells, and foreskin fibroblasts were reprogrammedinto induced pluripotent state using OCT4, SOX2, KLF4, and MYC genes.The iPSCs were found to be similar to embryonic stem cells in manycharacteristics, including DNA methylation patterns. Such concepts canbe used to reprogram cells, e.g., in combination with further circadiansynchronization techniques, described below.

In addition to inducement by action of immortalizing genes, cellsignaling was found to influence epigenetic processes governingdifferentiation. In the research of Baylin (e.g., Nature Biotechnology28 (10): 1033-8, 2010), several signaling pathways were suggested asimportant in the induction and maintenance of embryonic stem cells andin their differentiation. For example, signaling pathways of growthfactors can play a role in epigenetic regulation of cellulardifferentiation. These growth factors include, e.g., transforming growthfactors (TGFs), fibroblast growth factors (FGFs), and bone morphogeneticproteins. Another important factor in induction and differentiation canbe the Wnt signaling pathway.

Circadian rhythm influences on cellular synchronization can be employedin inducement of pluoripotential cells. For example, cells can bereprogrammed into inducible pluripotent stem cells using human clockgene and human Bmal1/2/3/4 genes and their E-box promoters. In oneaspect, fibroblast iPSCs can be generated by such reprogramming, andfurther directed to provide inducible neurons (iN), glial cells, orinducible neural progenitor cells (iNPCs), as desired. The reprogrammingfactor for each cell type is typically a transcriptional regulator thatis specific for the cellular lineage. Each factor can be modified to becontrolled by a circadian regulatory element (such as, E-box promotersor an artificial E-box-like promoter sequence tag). Such promotersequences can be added to each transcriptional regulator, thus forming anovel transcriptional element for control regulated by human Clock andBmal genes.

To complement the biosensor systems described herein, induced cells canbe cultured together in a “tissue” structure, e.g., in three dimensions.In this way, the cell to cell contacts of interest can be studied, e.g.,using penetrating imaging available in the form of near infraredreporter signals. For example, co-cultures of iN and iG can be preparedto create a 3D model of a neuronal structure. The structure can befurther controlled, e.g., using an appropriately structured scaffold,e.g., using materials not opaque to reporter signals, e.g., for confocalmicroscopic review.

Screening and Assay Methods Using Biosensors in Model Cells

The biosensor constructs, e.g., engineered into model cells of interest,can more predictably provide assay and screening results relevant tolife science research and study of clinical pathologies of interest.That is, the present targeted biosensors in appropriate cell types,differentiated to a particular phenotype, can provide models more likelyto anticipate a normal response in the modeled organism than, e.g., oldart xenotypic models.

The initial steps in preparing a model system can be to identify thecell type of interest and the signal to be detected. For example, tostudy certain neurological disease states, one may choose to target avoltage sensor to the plasma membrane, e.g., in model cellsdifferentiated from iPS cells. Alternately, one may elect to study ablood cell cancer using a hematopoietic blast induced to a model CMLcell and signaling from a calcium sensitive biosensor construct.Typically the biosensor constructs are transiently expressed in thecells using, e.g., a CMV constitutive promoter or a cell-type-specificpromoter.

Once the model system is established, the biosensor can be monitored ina single cell, or across an array of cells. For example, a cell can beexposed to a signaling agent to see if the cell type responds to thatagent, e.g., a cytokine or candidate small molecule bioactive agent.Optionally, the cell can be co-transfected with a second expressionconstruct of a peptide of interest (e.g., a tumor-associated antigen oroncogene) to monitor any influence of the external gene in a signalingpathway.

In other embodiments, the cells can be segregated into arrayscomplementary to low, medium, or high throughput assay formats. Forexample, cells can be dispensed into 96-well plates, onto a micro-wellarray, or a FACS sorter, for separate exposure to library members ofputative candidate agents. Such arrays can be reviewed suing standardfluorescent detection equipment. Optionally, the arrays can be reviewedphotographically with digital CCD based cameras. Changes in a signal,e.g., as compared to a positive or negative reference, can be flaggedfor additional characterization.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention. The inventive concepts disclosed herein includecertain biosensors sensitive to intracellular conditions, cellsexpressing the biosensors, and methods of monitoring the cells to detectintracellular changes, e.g., associated with an activity of anexternally applied agent.

Example 1 Voltage Sensor Construct

Presented is a voltage sensor for use in intracellular environments. Thevoltage sensor is a peptide construct featuring a combination ofinteracting structural features. The expressed peptide constructincludes a transmembrane domain, voltage sensing domain, and afluorescent reporter. The transmembrane can anchor the sensor to a lipidmembrane in a cell of choice. The voltage sensing domain can include,e.g., polar or ionic groups that are sensitive to changes in thesurrounding ionic environment or to a voltage potential across theanchoring membrane. The fluorescent reporter is a fluorescent peptidesequence, e.g., adapted to be sensitive to conformational changes inother domains of the construct.

The transmembrane domain typically includes hydrophobic amino acidresidues that interact with lipids of the membrane to anchor theconstruct. Further, the transmembrane domain can provide multipletransmembrane structures, together comprising an ion channel orvoltage-gated channel.

The voltage sensing domain of the exemplary embodiment includes thefollowing peptide sequence:

(SEQ ID NO: 1) MSSVRYEQREEPSMVNGNFGNTEEKVEIDGDVTAPPKAAPRKSESVKKVHWNDVDQGPNGKSEVEEEERIDIPEISGLWWGENEHGGDDGRMEVPATWWNKLRKVISPFVMSFGERVEGVVLIIVDEVLVIVDLSVTDKSSGATTAISSISLAISEFFLIDIILHIFVEGESQYFSSKLNIFDAAIVIVTLLVTLVYTVLDAFTDFSGATNIPRMVNFLRTLRIIRLVRIIILVR ILRLASQKTISQN.

Structural features of note in the voltage sensing sequence include alow complexity region (amino acids 63-75), transmembrane domain (aminoacids 109-131), and ion transport (amino acids 149-245). Particularlyimportant residues in the conformational response action include I123,R220, R226, R229, R235, R238; particularly residues R235 and I123.

In this construct, the far-red long wavelength properties fluorophorered color intensities should be particularly sensitive to any changes inresidues R235 and I123. Conformational changes in the sensing andtransmembrane domains (e.g., particularly in a membrane anchoredenvironment) lead to substantial measurable changes in the intensity(ΔF) and spectrum contours of the fluorophore domain emissions.

The following is a sample of a useful fluorescent-red reporter domainsequence:

(SEQ ID NO: 2) MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHQFKCTGEGEGRPYEAFQTAKLKVTKGGPLPFAWDILSPQFMYGSRAFIKHPAGIPDFFKQSFPEGFTWERVTRYEDGGVVTVMQDTSLED.

Example 2 Calcium Sensor Construct

The structural features of exemplary calcium sensor constructs include acomplementary combination of a calmodulin-binding domain, a troponindomain, and a fluorescent reporter domain. The construct can furtherinclude one or more tag sequences to target the construct to aparticular intracellular location or environment. The complementarycombination of tandem arrays of EF-hand domains that include acalmodulin-like-binding domain and a troponin-like domain, and afluorescent reporter domain. The fluorescent reporter is a fluorescentpeptide sequence, e.g., adapted to be sensitive to conformationalchanges in other domains of the construct.

The calcium sensor components interact as follows. Calcium-binding to EFhand domains leads to a conformational change and surface hydrophobicitychanges in the peptide construct. The changed calcium binding domainsthen interact differently with the fluorescent reporter domain causing asubstantial and measurable change in the intensity of the fluorophore(ΔF) emissions.

The calcium sensing domains of the exemplary embodiment includes thefollowing peptide sequence:

(SEQ ID NO: 12) MVDSSRRKWNKAGHAVRAIGRLSSPVVSERMYPEDGALKSEIKKGLRLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQCERAEGRHSTGGMDELYKGGTGGSLVSKGEENNMAVIKAEFMRFKEHMEAGSVNGHEFEIAEGEGEGRPYEAGTQTARLKVTKGGPLPFAWDAILSPQIMYGSAKAYVKHPADIAPDYLKLSFPEAGFKWERVMNFEDGGVVHVNQADSSLQDGVFIAYKVKLRGTNFAPPDGPVMQKKATMGWEATRDQLTEEEFRASFNHFDRDHSGTLGPEEFKACLISLDHMVLLTTKELGTVMRSLGQNPTEAELQDMINEVDADGDGTHOPEFLTMMARKMMNDTDSEEEGVQGTSEEEELANCFRIFDKDANGFIDEELGEILRATGEHVTEEDIEDLMKDSDKNNGRIDFGEKLTDEEVFKEAFSLFDKDGDGTITTKELGTVMRSLELDAIIEEVDEDGSGTIDFEEFLVMMVRQGQNPTKEEELANCFRIFDKN ADGFIDIEELGEILRAT.

The red fluorescent indicator can include the following far-red reporterdomain sequence:

(SEQ ID NO: 6) MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHQFKCTGEGEGRPYEAFQTAKLKVTKGGPLPFAWDILSPQFMYGSRAFIKHPAGIPDFFKQSFPEGFTWERVTRYEDGGVVTVMQDTSLED.

Example 3 Targeting Tags

In many cases, it is advantageous to direct the voltage sensor orcalcium sensor to a particular intracellular membrane or compartment.The biosensors of the invention can include peptide segments adapted tohave an affinity for a cellular target.

Exemplary peptide sequences useful in targeting biosensors to a desiredintracellular location include, e.g.:

NLS tag (protein sequence): (SEQ ID NO: 3) DPKKKRKV. ER tag:(SEQ ID NO: 8) KDEL  Endosome tag: (SEQ ID NO: 9) NPTY--DXXLL--YXXoo(protein sequence; tandem motifs; oo = 2two residues with hydrophobic side groups; Ciliary tag:VxPx-RVxP-KVHPSST-AxEGG (protein sequence; tandem motifs-SEQ ID NO: 10)Human endosome sequence tag (SEQ ID NO: 11)MTSRKKVLLKVIILGDSGVGKTSLMHRYVND  SEQ ID NO: 16 human synapsin tag:CCTGCAGGGCCCACTAGTATCTGCAGAGGGCCCTGCGTATGAGTGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGTGGGGGTGCCTACCTGACGACCGACCCCGACCCACTGGACAAGCACCCAACCCCCATTCCCCAAATTGCGCATCCCCTATCAGAGAGGGGGAGGGGAAACAGGATGCGGCGAGGCGCGTGCGCACTGCCAGCTTCAGCACCGCGGACAGTGCCTTCGCCCCCGCCTGGCGGCGCGCGCCACCGCCGCCTCAGCACTGAAGGCGCGCTGACGTCACTCGCCGGTCCCCCGCAAACTCCCCTTCCCGGCCACCTTGGTCGCGTCCGCGCCGCCGCCGGCCCAGCCGGACCGCACCACGCGAGGCGCGAGATAGGGGGGCACGGGCGCGACCATCTGCGCTGCGGCGCCGGCGACTCAGCGCTGCCTCAGTCTGCGGTGGGCAGCGGAGGAGTCGTGTCGTGCCTGAGAGCGCAGCTGTGCTCCTGGGCACCGCGCAGTCCGCCCCCGCGGCTCCTGGCCAGACCACCCCTAGGACCCCCTGCCC CAAGTCGCAGCCSEQ ID NO: 17 Human synaptic vesicle tag:MDCLCIVTTK KYRYQDEDTP PLEHSPAHLP NQANSPPVIVNTDT LEAPGYELQVNGTEGE MEYFEITLER GNSGLGFSIAGGTDNPHIG D DPSIFITKIIPGGAAAQDGR LRVNDSILFVNEVDBREBTH SAAV EALKEA GSIVRLYVMRRKPPAEKVMEIKLIKGPKGL GFSIAGGVGN STSLEITASMTempoEndo protein seq; TempoVol with Endosome tag (SEQ ID NO: 13)MTSRKKVLLKVIILGDSGVGKTSLMHRYVNDMSSVRYEQREEPSMVNGNFGNTEEKVEIDGDVTAPPKAAPRKSESVKKVHWNDVDQGPNGKSEVRNEERIDIPEISALWWGENEHGADDGRMELPATMWNKLRKVISPFVMSFGERVEGVVLIIVDEVLVIVDLSVTDKSSNATTAIESISLAISFFFLIDIILRIFVEGFNQYFSSKLNIFDAAIVIVTLLVTLVYTVLDAFTDFSGATNIPRMVNFLRTLRIIRLVRIERL ASQKRELRLASRRTISQN.Tags are (in most cases) N-terminal for each sensor-reporter construct.

Example 4 Methods of Reprogramming Cells

Human fibroblasts can be reprogrammed to a pluripotent state. Such cellscan then be differentiated to a degree to provide cell type specificmodel systems for expression of the biosensors.

Fibroblast cells are reprogrammed into inducible pluripotent stem cells(iPSC) over the course of ˜10 days, using a circadian rhythm (cellularsynchronization) induced method in combination with transcriptionalregulatory control modification. For example, human clock gene and humanBmal1/2/3/4 genes can be employed, e.g., using associated E-boxpromoters.

Each reprogramming factor is typically a transcriptional regulator thatis specific for the cellular lineage and each factor is modified to becontrolled by a circadian regulatory element (E-box promoters or anartificial E-box-like promoter sequence tag). The promoter sequences areadded to each transcriptional regulator, thus forming a noveltranscriptional element and control, regulated by human Clock and Bmalgenes.

Fibroblast cells can be reprogrammed into inducible neurons (iN), glialcells (astrocytes included, iG), and inducible neural progenitor cells(iNPCs) using a similar approach as the iPSC but with specifictranscription factors suitable for their lineages.

Co-cultures of iN and iG are performed to create a 3D model, using aproprietary in vitro scaffold.

Example 5 Methods for Screening Reprogrammed Cells

The sensors described herein can be introduced into reprogrammed cells,e.g., to monitor interactions of signaling moieties intracellularly.Further, the reprogrammed cells can be exposed to candidate agentmolecules to monitor the effects of the agents on intracellular calciumlevels and/or voltage potential of the cells.

Nucleic acids encoding the biosensors can be transiently (48 hrs)transduced into selected cells for expression, e.g., using standardelectroporation techniques. Further genetic modifications can beperformed on the cells to express genes-of-interest (a GPCR, channel,receptor, etc). Such cells can act as representative models for study ofsignaling interactions and bioactive agents.

In one option, cells are initially directly derived from patientsamples. After reprogramming and introduction of biosensors, such cellscan provide model systems and assay results more likely to reflectpatient responses. In the patient derived cell context, e.g., patientfibroblasts can be rendered pluripotent, differentiated to the extentdesired to reflect, e.g., a target cell of a pathology. Depending on thepathology of interest, a biosensor is adapted to be targeted to alocation of interest, to detect relevant voltage or calcium levels, andprovide a signal appropriate to the detection system in use.

In another option, an external (e.g., non-endogenous) gene of interestis introduced to the cells for study. For example, a gene of interest,such as a receptor molecule, GPCR, or a channel, can be molecularlycloned from a human cDNA library before the gene and transfected intothe cells. A biosensor is also introduced to the cells, and a cellularfunctional assay is performed. In this way, encoded peptides of the genecan be studies in the unique environment of relevant cells. Such modelcells (e.g., reprogrammed human cells expressing abnormal receptors) canprovide biosensor signals more likely to reflect associated live animalresults than other in vitro assays.

The following is a typical experimental design. Cells of interest areseeded into a multi-well format (12, 24, 48, 96, 384, or 1538-wellformats). Sensor expression constructs are introduced using standardtransfection procedures. 36-48 hours post transfection, cellularfunctional assays may begin, after correctly assessing the expressionlevels of the sensors. Stimuli for each experiment depends on the natureof the receptor or channel being assayed and what are the relevantphenotypic/disease conditions applied. If an agent of interest (e.g., aparticular molecule or ligand) is known, it is dissolved in liquidsolution to be applied to the cells.

A baseline can be established before application of the agent. Typicallyexcitation wavelengths are set to ˜450-570 nm. Emission wavelengthstypically range ˜500-700 nm. Fluorescence is sampled every ˜3 seconds atan exposure time ˜500 ms. ΔF/F is calculated by subtracting the baselinefluorescence level (e.g., 20 data points), subtracted from the actualfluorescence response and normalized to the starting fluorescence value.The percent fluorescence change is calculated for each region ofinterest in a cell or tissue using scripts in ImageJ, Matlab or anyproprietary softwares developed by manufacturers (such as MolecularDevices, Tecan, BioTek, etc.) software algorithms.

Example 6 Screening and Discovery Using Human Cellular Models

It is envisioned that the biosensors and model cell systems describedherein can be used in any number of formats and contexts to monitorintercellular interactions, signal transduction, and intracellularresponses. For example, the following utilities can be realized.

1) Human iPSCs-derived can be employed to cell types to generate 2D or3D cellular models of human disease models and using biosensors toscreen for chemical, biologics, drug-like, and toxicology compounds,e.g., to identify and evaluate candidate drug compounds.

2) A synthesized and genetically encoded biosensor can be prepared tofunction as a reporter of Ca²⁺ concentrations in human cells, e.g.,resolving events in cellular compartments such as the nucleus,cytoplasm, plasma membrane, and/or the like.

3) A synthesized and genetically encoded biosensor can function as areporter of cellular voltage changes in human cells, e.g., incompartments such as the nucleus, cytoplasm, or plasma membrane. Voltagesensors could evaluate and report fluctuations in membrane potentialsdue to changes in sodium (Na⁺) and potassium (K⁺) concentrations.

4) Biosensors can be used in a cell-type specific manner. For example,based on the disclosures herein, one can genetically modify a biosensorto contain specific promoter sequences for cell types, e.g.,dopaminergic neurons, GABAergic neurons, astrocytes, cardiomyocytes,HSCs, NPCs, MSCs, cancer cells, cancer stem cells, and/or the like.Human and mammalian cell types are generally of particular importanceand interest.

5) Biosensors signaling in long wavelength ranges (e.g., 500 nm-750 nmrange) can be used to detect interactions through three dimensions of abiologic sample.

6) Using biosensors with sequence identities with at least 70% identityto the following sequences (to be shown soon).

7) Using human cellular models conveniently derived from fibroblast orblood to provide information specific to a patient or cell type.

8) Reprogramming Parkinson patient-derived inducible pluripotent cells(iPSCs) from blood or fibroblast biopsies are into neural progenitorcells (NPCs). Appropriate biosensors are introduced to the cells usingelectroporation or chemical transfection methods (standard). Plate cellsinto multi-well plates (24, 48, 95, or 1500-well plates). Applycompounds to the cells via standard loading methods. Fluorescenceexcitation begins the experiment. Stimulation solutions are followed bywashing solutions. Measurements are taken and changes in fluorescence(ΔF) and ratio (ΔF/F) are calculated and plotted against time(milli-seconds to seconds timeframe). Alternatively, to calculate usinga single intensiometric reporter would require standardization of thebaseline responses and calculation of the changes in fluorescentintensities in response to a chemical or biological compound.

9) Rett syndrome patient-derived inducible pluripotent cells (iPSCs)from blood or fibroblast biopsies are induced into primary neurons,astrocytes and co-cultures of neuronastrocytes from affected-patientsand unaffected individuals. Biosensors are introduced to the cells usingelectroporation or chemical transfection methods (standard). Cells areplated into multi-well plates (24, 48, 95, or 1500-well plates).Compounds are applied to the cells via standard loading methods.Fluorescence excitation begins the experiment. Stimulation solutions arefollowed by washing solutions. Measurements are taken and changes influorescence (ΔF) and ratio (ΔF/F) are calculated and plotted againsttime (milli-seconds to seconds timeframe). Alternatively, to calculateusing a single intensiometric reporter would require standardization ofthe baseline responses and calculation of the changes in fluorescentintensities in response to a chemical or biological compound.

10) An early-to-mid-stage biopharmaceutical company looking for a newmethod of screening their library of compounds (specifically, biologicsand synthesized peptides) requires a new screening method. Progenitorcells (NPCs, MSCs, HSCs) are generated and voltage-biosensors areintroduced to the cells. Then, cells are plated into multi-well plates(24, 48, 95, or 1500-well plates). Compounds are applied to the cellsvia standard loading methods. Fluorescence excitation begins theexperiment. Stimulation solutions are followed by washing solutions.Measurements are taken and changes in fluorescence (ΔF) and ratio (ΔF/F)are calculated and plotted against time (milli-seconds to secondstimeframe). Alternatively, to calculate using a single intensiometricreporter would require standardization of the baseline responses andcalculation of the changes in fluorescent intensities in response to achemical or biological compound.

11) A company wishes to adopt a new toxicology technology to evaluatethe effects of their candidate compounds in a physiologically relevantcellular environment, e.g., using human primary/derived/induced celltypes and co-cultures in a 2D or 3D model. Equipment would include amulti-plate fluorescent reader (example: TECAN's M1000Pro) or ahigh-content cellular imager (GE's IN Cell 6000).

12) A company wishes to screen a drug candidate against a population ofpatients' derived cells (personalized medicine indications), in order todetermine the most appropriate genotypes for the candidate compound (forclinical phase 1/2/3 studies and evaluation). The company could beoffered two sets of custom-services—first, to make progenitor cell types(NPCs, HPCs, or MSCs) and introduce biosensors for screening; second, tomake specific cell types (neurons, glial, cardiomyocytes, liver, etc)and introduce biosensors for screening.

Example 7 2D Versus 3D Human Cellular Models

Traditionally, cell culture models in 2D are performed as monolayers.Culturing human cells in 3D requires specialized cell culture disheswith coating (e.g., Lipidure® coating or Nunc®) and sometimes, U-shaped,V-shaped, or F-shaped dish bottoms. Recently, numerous studies in humancancer and stem cell fields have pointed to the importance of 3Dculture. 3D cultures allow cancer cells to form tumors as spheroids (seeFIG. 21. Many in the academic literature have suggested that the 3Dspheroids are more predictive of cancer cellular responses to drugcompounds. Thus, using 3D spheroid models provide a new and improvedmodel to predict tumor responses to a chemical compound (aka. chemicalcompound in a drug library).

Example 8 ATP Biosensor

ATP sensing is important to cellular function, especially in themitochondria. For mammalian cells, ATP measurements indicate metabolicfunctions, which may be indicative of dysfunctions due to disease statesdue to human mutations or polymorphisms, signal transduction pathwaydisruptions, or mitochondrial dysfunction. Thus, developing an ATPbiosensor as a cellular reporter is of critical importance. Tempo's ATPbiosensor includes a domain that senses and binds to ATP and a domainthat is a fluorescent reporter in the 605 nm to 635 nm(excitation/emission) range.

ATP-binding biosensor, TempoATP:

(SEQ ID NO: 14) MDYKDDDDKKTNWQKRIYRVKPCVICKVAPRDWWVENRHLRIYTMCKTCFSNCINYGDDTYYGHDDWLMYTDCKEFSNTYHNLGRLPDEDRHWSASCHHHHHHMGMSGSMVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTMRIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDEEKQSFPEGFTWERVTRYEDGGVVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTHMALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAVAKFAGLGGGMDELYK.

Example 9 Heme Biosensor

Heme sensing is important to cellular function, especially in thestudies of mitochondria function, as well as in cancer/tumor hypoxiastudies (need Refs?). For mammalian cells, cellular metabolic rate ismeasured by oxygen consumption and indicates metabolic functions.Dysfunctions and irregularities in the cellular models may be due todisease phenotypes in the cell (due to human mutations orpolymorphisms), signal transduction pathway disruptions, ormitochondrial dysfunction. Thus, developing a Heme biosensor as acellular reporter is of critical importance. Tempo's Heme biosensorincludes a domain that senses and binds to heme (amino acid residues1-205) and a domain that is a fluorescent reporter in the 605 nm to 635nm (excitation/emission) range.

Oxygen-heme-binding biosensor, TempoHEME

(SEQ ID NO: 15) MAAMLEPEPVVAEGTAAQAVETPDWEAPEDAGAQPGSYEIRHYGPAKWVSTCVESMDWDSAVQTGFTKLNSYIQGKNEKGMKIKMTAPVLSYVEPGPGPFSESTITISLYIPSEQQSDPPRPSESDVFIEDRAKMTVFARCFEGFCSAQKNQEQLLTLASILREEGKVFDEKVFYTAGYNSPFRLLDKNNEVWLIQKNKPFKANEMVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTMRIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGGVVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTHMALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAVAKFAGLGGGMDEL YK.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovecan be used in various combinations. All publications, patents, patentapplications, and/or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application,and/or other document were individually indicated to be incorporated byreference for all purposes.

What is claimed is:
 1. A calcium sensor peptide construct comprising: anEFhand domain at least 80% identical to the sequence of SEQ ID NO: 5; acalcium binding domain; and, a fluorescent reporter domain, wherein thefluorescent reporter is adapted to emit wavelengths in the range from500 nm to 750 nm; wherein the fluorescent reporter domain is adapted tochange fluorescent emissions characteristics in response toconformational changes in the calcium binding or EFhand domains.
 2. Thecalcium sensor peptide construct of claim 1, wherein the calcium bindingdomain comprises a calmodulin peptide sequence.
 3. The calcium sensorpeptide construct of claim 1, further including a calcium sensor domainat least 80% identical to: SEQ ID NO:
 12. 4. The calcium sensor peptideconstruct of claim 1, wherein the fluorescent reporter domain is atleast 90% identical to: SEQ ID NO:
 6. 5. A calcium sensor peptideconstruct comprising: an EFhand domain; a calcium binding domain atleast 80% identical to the sequence of SEQ ID NO: 4; and, a fluorescentreporter domain, wherein the fluorescent reporter is adapted to emitwavelengths in the range from 500 nm to 750 nm; wherein the fluorescentreporter domain is adapted to change fluorescent emissionscharacteristics in response to conformational changes in the calciumbinding or the EFhand domains.
 6. The calcium sensor peptide constructof claim 5 wherein the EFhand domain is at least 80% identical to thesequence of SEQ ID NO:
 5. 7. The calcium sensor peptide construct ofclaim 5, further including a calcium sensor domain at least 80%identical to SEQ ID NO:
 12. 8. The calcium sensor peptide construct ofclaim 5, wherein the fluorescent reporter domain is at least 90%identical to SEQ ID NO: 6.